MPRAnalyze: statistical framework for massively parallel reporter assays.
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ABSTRACT:
SUBMITTER: Ashuach T
PROVIDER: S-EPMC6717970 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Noncoding DNA contains gene regulatory elements that alter gene expression, and the function of these elements can be modified by genetic variation. Massively parallel reporter assays (MPRA) enable high-throughput identification and characterization of functional genetic variants, but the statistical methods to identify allelic effects in MPRA data have not been fully developed. In this study, we demonstrate how the baseline allelic imbalance in MPRA libraries can produce biased results, and we propose a novel, nonparametric, adaptive testing method that is robust to this bias. We compare the performance of this method with other commonly used methods, and we demonstrate that our novel adaptive method controls Type I error in a wide range of scenarios while maintaining excellent power. We have implemented these tests along with routines for simulating MPRA data in the Analysis Toolset for MPRA (@MPRA), an R package for the design and analyses of MPRA experiments. It is publicly available at http://github.com/redaq/atMPRA.
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Project description:A long-standing goal of evolutionary biology is to decode how gene regulation contributes to organismal diversity. Doing so is challenging because it is hard to predict function from non-coding sequence and to perform molecular research with non-model taxa. Massively parallel reporter assays (MPRAs) enable the testing of thousands to millions of sequences for regulatory activity simultaneously. Here, we discuss the execution, advantages, and limitations of MPRAs, with a focus on evolutionary questions. We propose solutions for extending MPRAs to rare taxa and those with limited genomic resources, and we underscore MPRA's broad potential for driving genome-scale, functional studies across organisms.
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Project description:In many human diseases, associated genetic changes tend to occur within noncoding regions, whose effect might be related to transcriptional control. A central goal in human genetics is to understand the function of such noncoding regions: given a region that is statistically associated with changes in gene expression (expression quantitative trait locus [eQTL]), does it in fact play a regulatory role? And if so, how is this role "coded" in its sequence? These questions were the subject of the Critical Assessment of Genome Interpretation eQTL challenge. Participants were given a set of sequences that flank eQTLs in humans and were asked to predict whether these are capable of regulating transcription (as evaluated by massively parallel reporter assays), and whether this capability changes between alternative alleles. Here, we report lessons learned from this community effort. By inspecting predictive properties in isolation, and conducting meta-analysis over the competing methods, we find that using chromatin accessibility and transcription factor binding as features in an ensemble of classifiers or regression models leads to the most accurate results. We then characterize the loci that are harder to predict, putting the spotlight on areas of weakness, which we expect to be the subject of future studies.
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Project description:Massively parallel reporter assay (MPRA) is a high-throughput method that enables the study of the regulatory activities of tens of thousands of DNA oligonucleotides in a single experiment. While MPRA experiments have grown in popularity, their small sample sizes compared to the scale of the human genome limits our understanding of the regulatory effects they detect. To address this, we develop a deep learning model, MpraNet, to distinguish potential MPRA targets from the background genome. This model achieves high discriminative performance (AUROC = 0.85) at differentiating MPRA positives from a set of control variants that mimic the background genome when applied to the lymphoblastoid cell line. We observe that existing functional scores represent very distinct functional effects, and most of them fail to characterize the regulatory effect that MPRA detects. Using MpraNet, we predict potential MPRA functional variants across the genome and identify the distributions of MPRA effect relative to other characteristics of genetic variation, including allele frequency, alternative functional annotations specified by FAVOR, and phenome-wide associations. We also observed that the predicted MPRA positives are not uniformly distributed across the genome; instead, they are clumped together in active regions comprising 9.95% of the genome and inactive regions comprising 89.07% of the genome. Furthermore, we propose our model as a screen to filter MPRA experiment candidates at genome-wide scale, enabling future experiments to be more cost-efficient by increasing precision relative to that observed from previous MPRAs.
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Project description:Motivation:The majority of the human genome is composed of non-coding regions containing regulatory elements such as enhancers, which are crucial for controlling gene expression. Many variants associated with complex traits are in these regions, and may disrupt gene regulatory sequences. Consequently, it is important to not only identify true enhancers but also to test if a variant within an enhancer affects gene regulation. Recently, allele-specific analysis in high-throughput reporter assays, such as massively parallel reporter assays (MPRAs), have been used to functionally validate non-coding variants. However, we are still missing high-quality and robust data analysis tools for these datasets. Results:We have further developed our method for allele-specific analysis QuASAR (quantitative allele-specific analysis of reads) to analyze allele-specific signals in barcoded read counts data from MPRA. Using this approach, we can take into account the uncertainty on the original plasmid proportions, over-dispersion, and sequencing errors. The provided allelic skew estimate and its standard error also simplifies meta-analysis of replicate experiments. Additionally, we show that a beta-binomial distribution better models the variability present in the allelic imbalance of these synthetic reporters and results in a test that is statistically well calibrated under the null. Applying this approach to the MPRA data, we found 602 SNPs with significant (false discovery rate 10%) allele-specific regulatory function in LCLs. We also show that we can combine MPRA with QuASAR estimates to validate existing experimental and computational annotations of regulatory variants. Our study shows that with appropriate data analysis tools, we can improve the power to detect allelic effects in high-throughput reporter assays. Availability and implementation:http://github.com/piquelab/QuASAR/tree/master/mpra. Contact:fluca@wayne.edu or rpique@wayne.edu. Supplementary information:Supplementary data are available online at Bioinformatics.
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Project description:Massively parallel reporter assays (MPRAs) are a high-throughput method for evaluating in vitro activities of thousands of candidate cis-regulatory elements (CREs). In these assays, candidate sequences are cloned upstream or downstream from a reporter gene tagged by unique DNA sequences. However, tag sequences may themselves affect reporter gene expression and lead to major potential biases in the measured cis-regulatory activity. Here, we present a sequence-based method for correcting tag-sequence-specific effects and show that our method can significantly reduce this source of variation and improve the identification of functional regulatory variants by MPRAs. We also show that our model captures sequence features associated with post-transcriptional regulation of mRNA. Thus, this new method helps not only to improve detection of regulatory signals in MPRA experiments but also to design better MPRA protocols.
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Project description:BackgroundMassively parallel reporter assays (MPRAs) have emerged as a popular means for understanding noncoding variation in a variety of conditions. While a large number of experiments have been described in the literature, analysis typically uses ad-hoc methods. There has been little attention to comparing performance of methods across datasets.ResultsWe present the mpralm method which we show is calibrated and powerful, by analyzing its performance on multiple MPRA datasets. We show that it outperforms existing statistical methods for analysis of this data type, in the first comprehensive evaluation of statistical methods on several datasets. We investigate theoretical and real-data properties of barcode summarization methods and show an unappreciated impact of summarization method for some datasets. Finally, we use our model to conduct a power analysis for this assay and show substantial improvements in power by performing up to 6 replicates per condition, whereas sequencing depth has smaller impact; we recommend to always use at least 4 replicates. An R package is available from the Bioconductor project.ConclusionsTogether, these results inform recommendations for differential analysis, general group comparisons, and power analysis and will help improve design and analysis of MPRA experiments.
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Project description:Gene regulatory elements play a key role in orchestrating gene expression during cellular differentiation, but what determines their function over time remains largely unknown. Here, we perform perturbation-based massively parallel reporter assays at seven early time points of neural differentiation to systematically characterize how regulatory elements and motifs within them guide cellular differentiation. By perturbing over 2,000 putative DNA binding motifs in active regulatory regions, we delineate four categories of functional elements, and observe that activity direction is mostly determined by the sequence itself, while the magnitude of effect depends on the cellular environment. We also find that fine-tuning transcription rates is often achieved by a combined activity of adjacent activating and repressing elements. Our work provides a blueprint for the sequence components needed to induce different transcriptional patterns in general and specifically during neural differentiation.
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Project description:Neuropsychiatric phenotypes have long been known to be influenced by heritable risk factors, directly confirmed by the past decade of genetic studies that have revealed specific genetic variants enriched in disease cohorts. However, the initial hope that a small set of genes would be responsible for a given disorder proved false. The more complex reality is that a given disorder may be influenced by myriad small-effect noncoding variants and/or by rare but severe coding variants, many de novo. Noncoding genomic sequences-for which molecular functions cannot usually be inferred-harbor a large portion of these variants, creating a substantial barrier to understanding higher-order molecular and biological systems of disease. Fortunately, novel genetic technologies-scalable oligonucleotide synthesis, RNA sequencing, and CRISPR (clustered regularly interspaced short palindromic repeats)-have opened novel avenues to experimentally identify biologically significant variants en masse. Massively parallel reporter assays (MPRAs) are an especially versatile technique resulting from such innovations. MPRAs are powerful molecular genetics tools that can be used to screen thousands of untranscribed or untranslated sequences and their variants for functional effects in a single experiment. This approach, though underutilized in psychiatric genetics, has several useful features for the field. We review methods for assaying putatively functional genetic variants and regions, emphasizing MPRAs and the opportunities they hold for dissection of psychiatric polygenicity. We discuss literature applying functional assays in neurogenetics, highlighting strengths, caveats, and design considerations-especially regarding disease-relevant variables (cell type, neurodevelopment, and sex), and we ultimately propose applications of MPRA to both computational and experimental neurogenetics of polygenic disease risk.
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Project description:Family and population studies indicate clear heritability of major depressive disorder (MDD), though its underlying biology remains unclear. The majority of single-nucleotide polymorphism (SNP) linkage blocks associated with MDD by genome-wide association studies (GWASes) are believed to alter transcriptional regulators (e.g., enhancers, promoters) based on enrichment of marks correlated with these functions. A key to understanding MDD pathophysiology will be elucidation of which SNPs are functional and how such functional variants biologically converge to elicit the disease. Furthermore, retinoids can elicit MDD in patients and promote depressive-like behaviors in rodent models, acting via a regulatory system of retinoid receptor transcription factors (TFs). We therefore sought to simultaneously identify functional genetic variants and assess retinoid pathway regulation of MDD risk loci. Using Massively Parallel Reporter Assays (MPRAs), we functionally screened over 1000 SNPs prioritized from 39 neuropsychiatric trait/disease GWAS loci, selecting SNPs based on overlap with predicted regulatory features-including expression quantitative trait loci (eQTL) and histone marks-from human brains and cell cultures. We identified >100 SNPs with allelic effects on expression in a retinoid-responsive model system. Functional SNPs were enriched for binding sequences of retinoic acid-receptive transcription factors (TFs), with additional allelic differences unmasked by treatment with all-trans retinoic acid (ATRA). Finally, motifs overrepresented across functional SNPs corresponded to TFs highly specific to serotonergic neurons, suggesting an in vivo site of action. Our application of MPRAs to screen MDD-associated SNPs suggests a shared transcriptional-regulatory program across loci, a component of which is unmasked by retinoids.
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